Plant necrotrophic bacterial disease resistance phenotypes, QTL, and metabolites identified through integrated genetic mapping and metabolomics in Solanum species.

Most food crops are susceptible to necrotrophic bacteria that cause rotting and wilting diseases in fleshy organs and foods. All varieties of cultivated potato (Solanum tuberosum L.) are susceptible to diseases caused by Pectobacterium species, but resistance has been demonstrated in wild potato relatives including S. chacoense. Previous studies demonstrated that resistance is in part mediated by antivirulence activity of phytochemicals in stems and tubers. Little is known about the genetic basis of antivirulence traits, and the potential for inheritance and introgression into cultivated potato is unclear. Here, the metabolites and genetic loci associated with antivirulence traits in S. chacoense were elucidated by screening a sequenced S. tuberosum x S. chacoense recombinant inbred line (RIL) population for antivirulence traits of its metabolite extracts. Metabolite extracts from the RILs exhibited a quantitative distribution for two antivirulence traits that were positively correlated: quorum sensing inhibition and exo-protease inhibition, with some evidence of transgressive segregation, supporting the role of multiple loci and metabolites regulating these resistance-associated systems. Metabolomics was performed on the highly resistant and susceptible RILs that revealed 30 metabolites associated with resistance, including several alkaloids and terpenes. Specifically, several prenylated metabolites were more abundant in resistant RILs. We constructed a high-density linkage map with 795 SNPs mapped to 12 linkage groups, spanning a length of 1,507 cM and a density of 1 marker per 1.89 cM. Genetic mapping of the antivirulence and metabolite data identified five quantitative trait loci (QTLs) related to quorum sensing inhibition that explained 8-28% of the phenotypic variation and two QTLs for protease activity inhibition that explained 14-19% of the phenotypic variation. Several candidate genes including alkaloid, and secondary metabolite biosynthesis that are related to disease resistance were identified within these QTLs. Taken together, these data support that quorum sensing inhibition and exo-protease inhibition assays may serve as breeding targets to improve resistance to nectrotrophic bacterial pathogens in potato and other plants. The identified candidate genes and metabolites can be utilized in marker assisted selection and genomic selection to improve soft- rot and blackleg disease resistance.

Multilocus sequence and phenotypic analysis of Pectobacterium and Dickeya type strains for identification of soft rot Pectobacteriaceae from symptomatic potato stems and tubers in Pennsylvania.

Outbreaks of potato blackleg and soft rot caused by Pectobacterium species and more recently Dickeya species across the U.S. mid-Atlantic region have caused yield loss due to poor emergence as well as losses from stem and tuber rot. To develop management strategies for soft rot diseases, we must first identify which members of the soft rot Pectobacteriaceae are present in regional potato plantings. However, the rapidly expanding number of soft rot Pectobacteriaceae species and the lack of readily available comparative data for type strains of Pectobacterium and Dickeya hinder quick identification. This manuscript provides a comparative analysis of soft rot Pectobacteriaceae and a comprehensive comparison of type strains from this group using rep-PCR, MLSA and 16S sequence analysis, as well as phenotypic and physiological analyses using Biolog GEN III plates. These data were used to identify isolates cultured from symptomatic potato stems collected between 2016 and 2018. The isolates were characterized for phenotypic traits and by sequence analysis to identify the bacteria from potatoes with blackleg and soft rot symptoms in Pennsylvania potato fields. In this survey, P. actinidiae, P. brasiliense, P. polonicum, P. polaris, P. punjabense, P. parmentieri, and P. versatile were identified from Pennsylvania for the first time. Importantly, the presence of P. actinidiae in Pennsylvania represents the first report of this organism in the U.S. As expected, P. carotorvorum and D. dianthicola were also isolated. In addition to a resource for future work studying the Dickeya and Pectobacterium associated with potato blackleg and soft rot, we provide recommendations for future surveys to monitor for quarantine or emerging soft rot Pectobacteriace regionally.

Genetic Diversity and Population Structure of Leptosphaeria biglobosa from the Winter Oilseed Rape Region in China.

Phoma stem canker (blackleg), caused by the fungi Leptosphaeria maculans (anamorph Phoma lingam) and L. biglobosa, is one of the most devastating diseases in oilseed rape (Brassica napus L.) production worldwide. However, the population structure and genetic variation of L. biglobosa populations in China have rarely been investigated. Here, a collection of 214 fungal strains of blackleg from China (including Shaanxi, Jiangxi, Hubei, Jiangsu, Chongqing, Sichuan, Guangxi, Guizhou, Hunan, and Henan) and Europe (France and Ukraine) was identified as L. biglobosa. Three simple sequence repeat (SSR) markers were developed to characterize their population structure. The results showed that the Nei's average gene diversity ranged from 0.6771 for the population from Jiangsu to 0.3009 for the population from Hunan. In addition, most of the genetic variability (96%) occurred within groups and there were only relatively small amounts among groups (4.0%) (FST = 0.043, p = 0.042 < 0.05). Pairwise population differentiation (FST) suggested that significant genetic differentiation was observed between different L. biglobosa populations. Bayesian and unweighted average method analysis revealed that these L. biglobosa strains were clustered into three branches, and three European strains were similar to those from eastern China. The pathogenicity assay showed that those in Group III were significantly more virulent than those in Group I (t = 2.69, p = 0.016). The study also showed that Group III was dominant in Chinese L. biglobosa populations, which provides new insights for the further study of population evolution and the management of this pathogen.

Pseudomonas PA14H7: Identification and Quantification of the 7-Hydroxytropolone Iron Complex as an Active Metabolite against Dickeya, the Causal Agent of Blackleg on the Potato Plant.

Soft rot Pectobacteriaceae (SRP), such as Pectobacterium and Dickeya, are phytopathogenic agents responsible for blackleg disease on several crops, such as potatoes, affecting the yield and depressing the seed production quality. However, neither conventional nor biocontrol products are available on the market to control this disease. In this study Pseudomonas PA14H7, a bacteria isolated from potato rhizosphere, was selected as a potential antagonist agent against Dickeya solani. In order to understand the mechanism involved in this antagonism, we managed to identify the main active molecule(s) produced by PA14H7. Cell-free supernatant (CFS) of PA14H7 cultures were extracted and analyzed using LC-MS, GC-MS, and NMR. We further correlated the biological activity against Dickeya solani of extracted CFS-PA14H7 to the presence of 7-hydroxytropolone (7-HT) complexed with iron. In a second time, we have synthesized this molecule and determined accurately using LC-UV, LC-MS, and GC-MS that, after 48 h incubation, PA14H7 released, in its CFS, around 9 mg/L of 7-HT. The biological activities of CFS-PA14H7 vs. synthetic 7-HT, at this concentration, were evaluated to have a similar bacteriostatic effect on the growth of Dickeya solani. Even if 7-HT is produced by other Pseudomonas species and is mostly known for its antibacterial and antifungal activities, this is the first description of its involvement as an effective molecule against pectinolytic bacteria. Our work opens the way for the comprehension of the mode of action of PA14H7 as a biocontrol agent against potato blackleg.

Effect of formalin percentage, incubation time and temperature on Clostridium chauvoei culture inactivation and immunogenicity.

OBJECTIVES: In order to find the optimal inactivation conditions for Clostridium chauvoei culture, different factors were investigated and the immunogenicity of inactivated cultures was studied. METHODS: C. chauvoei was cultured with different formalin percentages (0.3, 0.5 or 0.7% V/V), inactivation temperatures (37 °C or room temperature) and incubation times (one or two weeks). Sterility tests were performed and residual formaldehyde and pH were measured. Rabbits were immunized twice with inactivated cultures and sera were used for detection of immune response. RESULTS: In the one-week experiment, 0.5 and 0.7% formalin inactivated the bacteria after one week, and the percentage of 0.3 inactivated after three weeks. The residual formaldehyde at weeks 1 and 8 was not significantly different. In the two-week experiment, cultures treated with 0.3 and 0.5% formalin were inactivated after four weeks, and those with 0.7% formalin were inactivated after three weeks. Residual formaldehyde at week 8 differed significantly from that of week 1. Residual formaldehyde was affected by incubation temperature since it was lower at 37 °C than in room temperature. Also, a significant effect was observed for formalin on pH, as higher formalin contents led to lower pH values of the cultures. ELISA showed the lowest antibody titer achieved by 0.7% formalin group. Antibody titer was not different between 0.3 and 0.5% formalin. CONCLUSIONS: The best condition for inactivation of C. chauvoei was considered as one-week incubation with 0.5% formalin at 37 °C, leading to a high antibody response.

Screening for Fungicide Efficacy in Controlling Blackleg Disease in Wasabi (Eutrema japonicum).

Blackleg disease is devastating for wasabi (Eutrema japonicum) production, occurring at any time and everywhere within the main production area of the Sichuan Province, China. There have been very few studies on the chemical control of this disease. In this study, we isolated and identified a local popular strain of the pathogen Plenodomus wasabiae. The isolated fungus strain caused typical disease spots on the leaves and rhizomes upon inoculation back to wasabi seedlings. The symptoms of blackleg disease developed very quickly, becaming visible on the second day after exposure to P. wasabiae and leading to death within one week. We then evaluated the efficacy of ten widely used fungicides to screen out effective fungicides. The efficacy of the tested fungicides was determined through mycelial growth inhibition on medium plates. As a result, tebuconazole and pyraclostrobin were able to inhibit the mycelial growth of P. wasabiae, and the most widely used dimethomorph in local production areas produced the lowest inhibition activity (13.8%). Nevertheless, the highest control efficacy of tebuconazole and pyraclostrobin on wasabi seedlings was only 47.48% and 39.03%, respectively. Generally, the control efficacy of spraying the fungicide before inoculation was better than that after inoculation. An increase in the application concentration of the two fungicides did not proportionately result in improved performance. We cloned the full-length sequence of sterol 14-demethylase (CYP51) and cytochrome B (CYTB) of which the mutations may contribute to the possible antifungalresistance. These two genes of the isolated fungus do not possess any reported mutations that lead to fungicide resistance. Previous studies indicate that there is a significant difference between fungicides in terms of the effectiveness of controlling blackleg disease; however, the control efficacy of fungicides is limited in blackleg control. Therefore, field management to prevent wound infection and unfavorable environmental conditions are more important than pesticide management.

An Efficient Triplex TaqMan Quantitative PCR to Detect a Blackleg-Causing Lineage of Pectobacterium brasiliense in Potato Based on a Pangenome Analysis.

P. brasiliense is an important bacterial pathogen causing blackleg (BL) in potatoes. Nevertheless, P. brasiliense is often detected in seed lots that do not develop any of the typical blackleg symptoms in the potato crop when planted. Field bioassays identified that P. brasiliense strains can be categorized into two distinct classes, some able to cause blackleg symptoms and some unable to do it. A comparative pangenomic approach was performed on 116 P. brasiliense strains, of which 15 were characterized as BL-causing strains and 25 as non-causative. In a genetically homogeneous clade comprising all BL-causing P. brasiliense strains, two genes only present in the BL-causing strains were identified, one encoding a predicted lysozyme inhibitor Lprl (LZI) and one encoding a putative Toll/interleukin-1 receptor (TIR) domain-containing protein. TaqMan assays for the specific detection of BL-causing P. brasiliense were developed and integrated with the previously developed generic P. brasiliense assay into a triplex TaqMan assay. This simultaneous detection makes the scoring more efficient as only a single tube is needed, and it is more robust as BL-causing strains of P. brasiliense should be positive for all three assays. Individual P. brasiliense strains were found to be either positive for all three assays or only for the P. brasiliense assay. In potato samples, the mixed presence of BL-causing and not BL-causing P. brasiliense strains was observed as shown by the difference in Ct value of the TaqMan assays. However, upon extension of the number of strains, it became clear that in recent years additional BL-causing lineages of P. brasiliense were detected for which additional assays must be developed.

Comparative pangenome analyses provide insights into the evolution of Brassica rapa resistance gene analogues (RGAs).

Brassica rapa is grown worldwide as economically important vegetable and oilseed crop. However, its production is challenged by yield-limiting pathogens. The sustainable control of these pathogens mainly relies on the deployment of genetic resistance primarily driven by resistance gene analogues (RGAs). While several studies have identified RGAs in B. rapa, these were mainly based on a single genome reference and do not represent the full range of RGA diversity in B. rapa. In this study, we utilized the B. rapa pangenome, constructed from 71 lines encompassing 12 morphotypes, to describe a comprehensive repertoire of RGAs in B. rapa. We show that 309 RGAs were affected by presence-absence variation (PAV) and 223 RGAs were missing from the reference genome. The transmembrane leucine-rich repeat (TM-LRR) RGA class had more core gene types than variable genes, while the opposite was observed for nucleotide-binding site leucine-rich repeats (NLRs). Comparative analysis with the B. napus pangenome revealed significant RGA conservation (93%) between the two species. We identified 138 candidate RGAs located within known B. rapa disease resistance QTL, of which the majority were under negative selection. Using blackleg gene homologues, we demonstrated how these genes in B. napus were derived from B. rapa. This further clarifies the genetic relationship of these loci, which may be useful in narrowing-down candidate blackleg resistance genes. This study provides a novel genomic resource towards the identification of candidate genes for breeding disease resistance in B. rapa and its relatives.

Deep amplicon sequencing reveals extensive allelic diversity in the erg11/CYP51 promoter and allows multi-population DMI fungicide resistance monitoring in the canola pathogen Leptosphaeria maculans.

Continued use of fungicides provides a strong selection pressure towards strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11/CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established.

Dissimilar gene repertoires of Dickeya solani involved in the colonization of lesions and roots of Solanum tuberosum.

Dickeya and Pectobacterium species are necrotrophic pathogens that macerate stems (blackleg disease) and tubers (soft rot disease) of Solanum tuberosum. They proliferate by exploiting plant cell remains. They also colonize roots, even if no symptoms are observed. The genes involved in pre-symptomatic root colonization are poorly understood. Here, transposon-sequencing (Tn-seq) analysis of Dickeya solani living in macerated tissues revealed 126 genes important for competitive colonization of tuber lesions and 207 for stem lesions, including 96 genes common to both conditions. Common genes included acr genes involved in the detoxification of plant defense phytoalexins and kduD, kduI, eda (=kdgA), gudD, garK, garL, and garR genes involved in the assimilation of pectin and galactarate. In root colonization, Tn-seq highlighted 83 genes, all different from those in stem and tuber lesion conditions. They encode the exploitation of organic and mineral nutrients (dpp, ddp, dctA, and pst) including glucuronate (kdgK and yeiQ) and synthesis of metabolites: cellulose (celY and bcs), aryl polyene (ape), and oocydin (ooc). We constructed in-frame deletion mutants of bcsA, ddpA, apeH, and pstA genes. All mutants were virulent in stem infection assays, but they were impaired in the competitive colonization of roots. In addition, the ΔpstA mutant was impaired in its capacity to colonize progeny tubers. Overall, this work distinguished two metabolic networks supporting either an oligotrophic lifestyle on roots or a copiotrophic lifestyle in lesions. This work revealed novel traits and pathways important for understanding how the D. solani pathogen efficiently survives on roots, persists in the environment, and colonizes progeny tubers.

Prevalence of Cardiac Lesions in Cases of Bovine Blackleg in Tennessee (USA), 2004-2018.

Blackleg is a common cause of death in cattle, mostly caused by the bacterium Clostridium chauvoei. Cardiac lesions were traditionally considered uncommon in cases of blackleg in cattle until a 2018 study reported otherwise. This study was aimed at determining the prevalence of cardiac disease among cattle that died of blackleg in Tennessee, USA. The outcome of this study would reinforce the importance of assessing cardiac lesions in suspected cases of blackleg in cattle. The University of Tennessee Veterinary Medical Center database searched for cattle with a confirmed diagnosis of blackleg necropsied between 2004 and 2018. Of the 120 necropsy reports, 37 had a diagnosis of blackleg. Histology slides of skeletal muscle (26/37) and the heart (26/37) were reviewed to confirm the presence of supportive lesions. Of the 37 cases of blackleg identified, 26 animals (70.3%) had cardiac lesions, including 4 (10.8%) that had only cardiac involvement without skeletal muscle lesions. Specifically, (5.4%; 2/37) had only necrotizing myocarditis; (13.5%; 5/37) had only fibrinous or fibrinosuppurative pericarditis, epicarditis, or endocarditis; (51.4%; 19/26) had a combination of myocarditis and pericarditis, epicarditis, or endocarditis; and (29.7%; 11/37) had no lesions. Furthermore, of the 26 cases with cardiac lesions, 24 cases had gross lesions, while 2 cases were identified only by microscopic examination. This indicates that gross examination alone is insufficient to identify cardiac involvement in blackleg cases in cattle. Contrary to traditional perceptions, cardiac lesions in cases of bovine blackleg could be as high as 70% and are most often associated with skeletal muscle lesions. The prevalence of cardiac lesions in cases of blackleg in cattle may be higher when the heart is examined microscopically than if it is only evaluated grossly. Pathologists should specifically evaluate the heart for lesions in suspected cases of blackleg in cattle and utilize microscopic examination when gross lesions are absent.

First Report of Pectobacterium versatile Causing Diseases on Potato in Xinjiang Uygur Autonomous Region and Heilongjiang Province, China.

Pectobacterium spp. and Dickeya spp. cause aerial stem rot on potatoes worldwide (Charkowski, 2018). Potato plants (cv. Xisen6# or Youjin) with aerial stem rot or blackleg symptoms (Fig. S1) were observed in the commercial fields in Changji, Xinjiang Province in September 2021 and Harbin, Heilongjiang Province in August 2021, in China. The field disease incidences were 45-50% and 15-20% in Changji (2 ha) and Harbin (1 ha), respectively. Five diseased plants from each field site were collected to isolate the pathogen. Symptomatic stems were soaked in 75% ethanol for 2 min, rinsed, and ground in sterile distilled water (Handique et al. 2022). The suspension was plated onto a crystal violet pectate agar (CVP) plate (Ge et al. 2018). Three days after incubation at 28°C, bacterial colonies that developed pits on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al. 2002). Amplified 16S rDNA sequences from two isolates designated as ZRIMU1267 and ZRIMU1366 showed more than 99% sequence identity to P. versatile CFBP6051T type strain and the sequences were submitted to GenBank (accession numbers: OP476349, OP476350). Additionally, six housekeeping genes sequences were uploaded to GenBank: proA (OP487826, OP487832), gyrA (OP487828, OP487834), icdA (OP487823, OP487829), mdh (OP487825, OP487831), gapA (OP487824, OP487830), and rpoS (OP487827, OP487833) (Ma et al. 2007; Waleron et al. 2008). A phylogenetic tree based on concatenated sequences (MLSA) of the housekeeping genes (Fig. S2) of the two isolates was constructed using MEGA X (Tamura et al. 2013).The phylogenetic tree of MLSA sequences shows that the sequences from isolates ZRIMU1267 and ZRIMU1366 clustered with P. versatile CFBP6051T indicating that these isolates are P. versatile at the species level. Koch's postulate were performed on 3-week-old potato seedlings (cv. Favorita) and tubers. The bacterial suspension (100 µl, 105 CFU/ml) or sterile phosphate-buffered solution was injected into the crown area of the seedlings for the development of aerial stem rot or drenched in the potting mix for the development of blackleg, and the plants were covered with polybags to keep 100% humidity at 25° for 2 days. Five seedlings were inoculated for each strain and the experiment was repeated twice. Seven days after stem injection, the infected area of the inoculated seedlings was rotten, turned black, or even lodged, while the controls were symptomless (Fig. S3a). Four days after drench application, the seedlings were wilted and lodged, while the controls were symptomless (Fig. S3 c). Tuber slice assay for soft rot development was performed by adding bacterial suspension (100 µl, 105 CFU/ml). One day after inoculation, the infected tubers rotted, while the controls were symptomless (Fig. S3 b). ZRIMU1267 and ZRIMU1366 were reisolated from infected tissues on CVP plates and identified by 16S rRNA sequences to complete Koch's postulate. Diseases on potato has been reported to be caused by P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense in China (Zhao et al. 2018; Cao et al. 2021; Wang et al. 2021; Handique et al. 2022). P. versatile causing aerial stem rot on potatoes have been reported in Hebei province (Han et al. 2022), while our study reports P. versatile strains that are able to cause multiple diseases on potatoes in Xinjiang Uygur Autonomous Region and Heilongjiang provinces of China. The results indicate that P. versatile might be widely distributed in northern China, and it is necessary to include cropping season and post-harvest strategies to control diseases caused by P. versatile.

Increase of Glycoalkaloid Content in Potato Tubers by Greening as a Method to Reduce the Spread of Pectobacterium and Dickeya spp. in Seed Production Systems.

Dickeya and Pectobacterium species are the causal agents of blackleg and soft rot diseases. This article explores the possibility of using the glycoalkaloids (GAs) naturally produced by the potato tuber after the greening process as a blackleg control method. We first tested the effect of GAs extracted from four potato cultivars on the growth and viability of one Dickeya and one Pectobacterium strain in growth media. Then, four years of field experiments were performed in which the incidence of blackleg was assessed in plants grown from the seed tubers of cv. Agria that were subjected to various greening treatments. In the growth media, all GAs isolated from the four cultivars appeared to be bacteriostatic and bactericidal against both bacteria strains. The inhibitory effect varied among GAs from different cultivars. Except for a one-year field trial, the blackleg incidence was lower in plants grown from green seed tubers without the yield being affected. The blackleg control was marginal, probably due to the low production of GAs by the tubers of cv. Agria after greening. Based on our findings, seed tuber greening has a good potential for blackleg control after the identification of varieties that present optimal GA composition after greening.

Identification and characterization of opportunistic pathogen Pectobacterium polonicum causing potato blackleg in China.

Blackleg and aerial stem rot of potato (Solanum tuberosum L.), caused by soft rot enterobacteria of the genera Pectobacterium and Dickeya, has recently increased years in Hebei Province, China. Field surveys were performed during the 2021 potato growing season in Hebei to identify and characterize bacterial pathogens. Sixteen potato plants showing blackleg or aerial stem rot were collected from three potato-producing areas, and ten representative pectinolytic bacteria were isolated from symptomatic plants. 16S rDNA sequencing and multilocus sequence analysis were performed to determine the taxonomic position of the bacterial isolates. The isolates belonged to the genus Pectobacterium, including Pectobacterium atrosepticum, Pectobacterium carotovorum, Pectobacterium brasiliense, and Pectobacterium parmentieri. The exceptions were isolates BY21311 and BY21312, which belonged to a new species of Pectobacterium polonicum previously found in groundwater. The taxonomy of isolate BY21311 was confirmed using whole genome-based analysis. P. polonicum has only been identified in potato plants on one farm in Baoding region in China. Isolates BY21311 and BY21312 displayed similar physiological and biochemical traits to the type strain DPMP315T. Artificial inoculation assays revealed that isolate BY21311 fulfilled Koch's postulates for potato blackleg. These findings represent the first time P. polonicum, a water-associated Pectobacterium species may be the cause of blackleg in the field. Interestingly, P. polonicum BY21311 has reduced ability to macerate potato tubers when compared to P. atrosepticum, P. brasiliense, P. versatile, and P. parvum, which is more virulent in tubers than the type strain DPMP315T. The host range of isolate BY21311 was determined by injection method, which can impregnate five plants. Although the genome of isolate BY21311 harbors gene clusters encoding a type III secretion system, it did not elicit a hypersensitive response (HR) in Nicotiana benthamiana or N. tabacum leaves. T3SS effector AvrE and T4SS effector PilN were obtained by predicting isolate BY21311 genome. P. polonicum appears to show significant variations in gene content between two genomes, and gene content varies between isolates BY21311 and DPMP315T, with strain specific-genes involved in many aspects, including lipopolysaccharide biosynthesis, substrate translocation, T4SS and T6SS among others, suggesting that isolates BY21311 and DPMP315T might represent distinct clades within the species.

Identification of candidate genes for LepR1 resistance against Leptosphaeria maculans in Brassica napus.

Utilising resistance (R) genes, such as LepR1, against Leptosphaeria maculans, the causal agent of blackleg in canola (Brassica napus), could help manage the disease in the field and increase crop yield. Here we present a genome wide association study (GWAS) in B. napus to identify LepR1 candidate genes. Disease phenotyping of 104 B. napus genotypes revealed 30 resistant and 74 susceptible lines. Whole genome re-sequencing of these cultivars yielded over 3 million high quality single nucleotide polymorphisms (SNPs). GWAS in mixed linear model (MLM) revealed a total of 2,166 significant SNPs associated with LepR1 resistance. Of these SNPs, 2108 (97%) were found on chromosome A02 of B. napus cv. Darmor bzh v9 with a delineated LepR1_mlm1 QTL at 15.11-26.08 Mb. In LepR1_mlm1, there are 30 resistance gene analogs (RGAs) (13 nucleotide-binding site-leucine rich repeats (NLRs), 12 receptor-like kinases (RLKs), and 5 transmembrane-coiled-coil (TM-CCs)). Sequence analysis of alleles in resistant and susceptible lines was undertaken to identify candidate genes. This research provides insights into blackleg resistance in B. napus and assists identification of the functional LepR1 blackleg resistance gene.

First Report of Pectobacterium brasiliense Causing Bacterial Blackleg and Soft Rot of Potato in Pennsylvania.

Potato (Solanum tuberosum) plants showing blackleg and soft rot symptoms were collected at a commercial vegetable farm near Newmanstown, PA in August 2021 (Fig. S1). The incidence of potato blackleg in the unirrigated field was about 5 to 8%, but approximately 30% in the irrigated field. The diseased stems were cut into 5 cm and surface disinfected. The stem segments were placed into a 50-mL tube containing 15 mL of sterile water for 15 min for bacterial release. The bacterial suspension was streaked on crystal violet polypectate (CVP) (Hélias et al. 2012) plates and incubated at 28°C for 48 h. Three single colonies produced pits on CVP were picked and purified. Genomic DNA of all three isolates were extracted using the FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA). Polymerase chain reaction (PCR) was performed using all three extracted DNAs as a template with the primer pairs gapA 7F/938R (Cigna et al. 2017), recA F/R (Waleron et al. 2001), dnaA F/R (Schneider et al. 2011) and dnaX F/R (Slawiak et al. 2009) targeting the gapA, recA, dnaA and dnaX genes, respectively. Isolate 21PA01 was further studied as a representative isolate. PCR amplicons derived from both forward and reverse primers were sequenced and analyzed using the BLAST algorithm against the NCBI database (https://www.ncbi.nlm.nih.gov). The regions of gapA (GenBank accession No. ON989738), recA (ON989739), dnaA (OP121183), and dnaX (OP121184) had 99.86%, 100%, 98.88%, and 100% identities with Pectobacterium brasiliense strains S1.16.01.3M (MN167062.1), BL-2 (MW721598.1), IPO:4132 (CP059956.1), and BL-2 (MW721603.1), respectively. A phylogenetic maximum-likelihood tree of the concatenated genes with the length of 2551 bp was constructed to visualize the relationship among different species of Dickeya and Pectobacterium. As a result, 21PA01 was in a single monophyletic cluster with other Pectobacterium brasiliense reference strains (Fig. S2 C). To confirm the pathogen, Koch's postulates were performed. Seed pieces of potato 'Lamoka' were planted in potting mix in one-gallon plastic pots in a greenhouse. Three weeks after emergence, the stems of three plants were each injected with 10 µL of bacteria suspension of either 21PA01 at 107 CFU/mL, P. parmentieri ME175 in tryptic soy broth (TSB) at 107 CFU/mL or TSB at 2 cm above the soil line. Seven days after inoculation, stems inoculated with 21PA01 and ME175 showed black and rotten symptoms, whereas the TSB-injected control plants remained symptomless. In addition, 'Lamoka' tubers were inoculated by placing 10 µL 21PA01 and ME175 suspensions at 107 CFU/mL, and TSB in a 1-cm-deep hole poked in a tuber separately and then sealed with petroleum gel, followed by incubation in a moist chamber at 22 °C for 4 d. The 21PA01 and ME175 inoculated tubers showed soft rot symptoms, but the TSB treatment had no symptoms. Bacterial colonies were isolated from the infected stems and confirmed by the DNA sequences as described above. PCR result was negative on control plant samples. Both stem and tuber inoculation trials were repeated two times, and the results were consistent. Thus, 21PA01 was identified as Pectobacterium brasiliense. To our knowledge, this is the first report of P. brasiliense infecting potatoes in Pennsylvania, USA, although it has been reported somewhere else (van der Merwe et al. 2010, Zhao et al. 2018). This could be a new species in Northeastern US.

Development of a Duplex qPCR System for Detection and Quantification of the Two Canola Blackleg Pathogens Leptosphaeria biglobosa and L. maculans.

Two probe-based qPCR systems, namely P-Lb and P-Lm, specific to the canola blackleg pathogens Leptosphaeria biglobosa and L. maculans, respectively, were developed, and their efficiencies were tested. Each of the two systems targets a single-copy gene exclusively present in the corresponding species. The specificities of the two systems on the species level and their ubiquities on the subspecies level were confirmed by in silico sequence analyses and testing on L. biglobosa (17 strains), L. maculans (10 strains), and other plant pathogens (31 species). For sensitivities, the two systems were tested on synthesized DNA fragments (gBlock) of the targeted regions, from which a standard curve was generated for each system. In addition, standard curves were also generated on gBlocks for duplex qPCR in which the two systems were used in the same reaction. The two systems were further tested in both singleplex and duplex qPCR on DNA samples extracted from fungal spores, inoculated canola cotyledons, and naturally infected canola stubble samples collected from commercial fields. Our data indicated that the two systems are specific to L. biglobosa and L. maculans, respectively, and one reaction could detect as few as 200 spores of either species. When used in duplex qPCR on DNA samples with various origins, the two systems generated similar results as in singleplex qPCR. The duplex qPCR system, along with the sample preparation and DNA extraction specified in this study, constituted a first-reported duplex qPCR protocol for detection and quantification of the two blackleg pathogens from field samples.

Blackleg Yield Losses and Interactions with Verticillium Stripe in Canola (Brassica napus) in Canada.

Blackleg, caused by Leptosphaeria maculans, is an important disease of canola (Brassica napus). The pathogen can attack stems, leaves and pods, but basal stem cankers are most damaging and can result in significant yield losses. In Canada, Verticillium stripe (Verticillium longisporum) has recently emerged as another disease threat to canola. Symptoms of Verticillium stripe can resemble those of blackleg, and the two diseases may occur together. The effect of blackleg on yield was explored in field experiments with two canola hybrids and by evaluating a wider variety of hybrids in commercial crops in central Alberta, Canada. The impact on yield of L. maculans/V. longisporum interactions was also assessed under field and greenhouse conditions. In most hybrids, the relationship between blackleg severity and yield components was best explained by second-degree quadratic equations, although a linear relationship was found for one variety sampled in commercial fields. When L. maculans was co-inoculated with V. longisporum, blackleg severity and yield losses increased. In some cases, Verticillium stripe caused greater yield losses than blackleg. The results suggest that the interaction between L. maculans/V. longisporum may cause more severe losses in canola, highlighting the need for proactive disease management strategies.

Genome Sequence Resource of Pectobacterium polaris QK413-1 that Causes Blackleg on Potato in Fujian Province, China.

The Pectobacterium pathogens cause soft rot and blackleg diseases on many plants and crops, including potatoes. Here, we first report a high-quality genome assembly and announcement of the P. polaris strain QK413-1, which causes blackleg disease in potatoes in China. The QK413-1 genome was sequenced and assembled using the PacBio Sequel II and Illumina sequencing platform. The assembled genome has a total size of 5,005,507 bp with a GC content of 51.81%, encoding 4,782 open reading frames, including 639 virulence genes, 273 drug resistance genes, and 416 secreted proteins. The QK413-1 genome sequence provides a valuable resource for the control of potato blackleg and research into its mechanism.